Chemiluminescence and Bioluminescence in Nature

Bioluminescence is a scientific phenomenon that is complex in character because luminous organisms possess unique light-producing chemical reactions and have varied methods of controlling light. Luminous organisms do not fall under order of animals but are unevenly distributed across multiple animal orders. Furthermore, luminous organisms are often adapted to specialized environments and can be difficult to physically research. This paper will cover the background of bioluminescence, luminous reactions and their regulators, and the practical applications of knowledge in this field. The purpose of the research is to gain an adequate understanding of chemiluminescence in nature in order to predict the nature of future research and gauge its potential in the modern world. Research for this paper was accomplished through the reviewing of published scientific papers and literature on the subject. Some of the results reached include that bioluminescence is different in terrestrial and aquatic organisms and that a wide range of techniques are used to moderate light in both habitats. The conclusions that have been reached are that research of bioluminescence will surely accelerate and that further research of bioluminescence has potential in the areas of evolutionary biology, lighting technology, and medicine.

Introduction

When Christopher Columbus embarked on his voyage in 1492, he had to overcome many strong prejudices rooted in European folklore. Tales of sea monsters have captured the minds of Europeans for centuries, and Columbus was utterly mesmerized when the waters surrounding his ship began to shine. The alluring glow of bioluminescent organisms has continued to perplex humans all the way through modern times. Although science has illuminated the surprisingly dark realm of bioluminescence, further research is still required. This paper will cover the background, reactions, and uses of this complex, yet common phenomenon to determine the nature of future research and its applicability in other areas of science.

HISTORY

During the seventeenth century, the English physicist Robert Boyle conducted an important experiment concerning bioluminescence. Robert Boyle encased a piece of glowing wood within a glass bell and then proceeded to suck the air out the enclosure. As he took out the air, he observed that the glowing of the wood grew fainter and eventually was extinguished completely (Simon 114). This was scientific experiment demonstrated a principle concept of bioluminescence: oxygen plays a key role in luminous reactions. Towards the end of the nineteenth century, the French physiologist Dubois began to research luminous organisms in his marine laboratory. His major contribution was his experiment involving the Pholas clams. In his experiment, Dubois prepared two different solutions of clam juice. For the first solution, he mixed the clam juice with cool water and observed it glow for a while before it faded. He then mixed clam juice with hot water, but this solution failed to produce any light at all. In a stroke of genius, he decided to mix the two nonluminous solutions together. As soon as they were combined, the bluish light characteristic of the Pholas shone forth once again. This led Dubois to assume that in the cold solution, one substance was exhausted after luminescence and that in the hot solution, a different substance was destroyed. Thus, Dubois concluded that the unknown substance destroyed by the hot water was almost certainly an enzyme. An enzyme is an organic catalyst. Dubois identified that bioluminescence required an oxidizable substrate, an enzyme, and oxygen. He named the substrate “luciferin” and the enzyme “luciferase” (Simon 116). This major advancement was the stepping-stone into future research on this perplexing phenomenon.

FUNCTION

Although terrestrial habitats seem to be devoid of bacterial modes of light-production, there are a few cases of bacterial luminescence on land. Many supposed bioluminescent organisms such as mole crickets do not produce light themselves, but have turned out to be infected with parasitic forms of luminous bacteria. Luminous bacteria multiply within the hemolymph of arthropods (which is analogous to human blood cells). The infected creatures end up eventually end up dying (Nealson and Hasting 508). For example, the luminous bacteria X. luminescens live in the gut of a certain nematode belonging to the genus Heterorhabditis. Farmers greatly value nematodes because these un-segmented roundworms parasitize pest insects. The Heterorhabditis nematode enters the body of a host caterpillar through orifices like spiracles or the mouth. Once inside the caterpillar’s body, the nematode will proceed to penetrate the caterpillar’s hemocoel, the area containing hemolymph. When in contact with the hemolymph, the nematode will then release its fertilized eggs along with the bacteria X. luminescens. The bacteria then multiply and produce extracellular chitinase and lipases that the nematode uses to complete the its life cycle. X. luminescens also produces antibiotics that arrests the growth of bacteria that would otherwise outcompete it and also prevents the caterpillar from putrefying (Havens 1). It is interesting to note that the bacteria only glow while in the hemolymph of the caterpillar, but not inside the nematodes themselves. This land bacterium uses a biochemical reaction very similar to its marine counterparts. The overall general reaction is the same: the flavin-mononucleotide and long-chain aldehyde (fatty aldehyde) are oxidized in the presence of luciferase to produce water and light. FMNH2 + RCHO + O2 → FMN + H2O + RCOOH + Light (Havens 1).

FUNCTION

The most important luminous bacteria are the commensal forms that thrive inside the gut tracts of marine animals. It’s not unusual to find 5Ã-106 to 5Ã-107 colony-forming units of luminous bacteria per meter of intestinal surface (Nealson and Hastings 508). Colony-forming units are used in the area of microbiology to express quantities of viable bacteria capable of forming colonies or clusters visible to the human eye. This relationship between the bacteria and the host organisms seems to be commensal because luminous bacteria produce the enzyme chitinase, thereby benefiting their host if they eat marine crustaceans (a regular staple of marine diet). However, studies of the senorita fish Oxyjulis californica, the blacksmith fish Chrormis puntipinnis, and the half-naked hatchetfish Argyropelecus hemigymnus show that the occurrence of each fish was connected with the species composition of the planktonic luminous bacteria population (Nealson and Hastings 508). Fecal pellets were luminescent and contained colony-forming units of luminous bacteria. Similarly, luminous fecal pellets have been reported from the Antarctic cod and a species of midwater shrimp (Nealson and Hastings 508). Because the fecal pellets of these marine animals contain viable bacteria populations, it is possible that luminous bacteria mutually benefit through unintentional propagation by their host organisms.

Historical accounts from 19th century battlefield hospitals have shown that luminous bacteria in the open wounds of soldiers were considered to be a sign of healing (Nealson and Hastings 507). It is interesting to note that Xenorhabdus is known to produce antibiotics (Nealson and Hastings 508).

TYPES

Bioluminescence can be divided into two subcategories: terrestrial forms and aquatic forms. Terrestrial forms of bioluminescence are sparse and restricted to insects and their relatives. Beetles in particular have unique chemical reactions. Non-insect relatives include certain centipedes. Luminous centipedes are unique in that they secrete luminous slime (Simon 57). Land is largely devoid of luminous animals that utilize bacterial forms of light production. Luminous land animals are usually found in humid, heavily forested environments.

Contrarily, luminous bacteria dominate the majority of aquatic environments. Luminous bacteria even thrive in arctic waters. Luminous bacteria can exist as free-living bacteria, saprophytes, and as symbionts in relationships with various marine animals.

Luminous bacteria in their free-living forms are regularly present in seawater. Recent studies give further insights on luminous bacteria demography. A sampling of the waters off the coast of San Diego, California showed that Beneckea were common in the winter while P. fischeri was prevalent during the summer (Nealson and Hastings 505). A study of luminous bacteria depth distribution demonstrated that P. phosphoreum were most abundant in the midwater layer of the open ocean.

Saprophytic forms of bacteria are also extremely common. These forms of luminous bacteria are quite common and live on the surfaces of dead organic material. In fact, researchers often swab the outer surface of freshly killed fish or squid to start a culture of luminous bacteria.

BACTERIA REACTION

As opposed to most terrestrial forms of bioluminescence, bacterial bioluminescence is the dominant form in marine habitats. Currently, six species of marine luminous bacteria belonging to the genera of Photobacterium and Beneckea have been identified. There is one species of freshwater luminous bacteria (Vibrio). Like all forms of bioluminescence known to man, light of bacterial origin involves a luciferin-luciferase reaction. Luminous bacteria generate light through the luciferase-catalyzed oxidation of the substrate flavin-mononucleotide (FMNH2) with the associated oxidation of a long-chain aldehyde. What is unique about this reaction is that it is very slow; it takes ten seconds for a single luciferase cycle to occur, making it one of the slowest enzymes (Nealson and Hastings 497). Luciferases from various luminous bacteria have been isolated; although they all share high specifity for flavin-mononucleotide and long-aldehyde, the luciferase of Photobacteria exhibits fast decay while that of Benecka exhibits slow decay. Recent amino acid sequencing of P. fischeri and B. harveyi support the theory that the luciferases of these two species evolved from the same monomer. Bacterial luminescence has high oxygen affinity and occurs under low concentrations of oxygen or microaerophillic conditions. It is also interesting to note that facultative anaerobes, produce extracellular chitinase, and have specific requirements for sodium ion (Nealson and Hastings 497).

FIREFLY REACTION

The most widely known example of bioluminescence is in the fireflies. Bioluminescence in members of the beetle order is very unique. Fireflies use precisely timed light signals to attract mates. Specialized cells within the lantern section of the abdomen like all forms of bioluminescence involve a luciferin-luciferase reaction. This reaction can be divided into two steps. First, luciferin combines with adenosine triphosphate (ATP) to form luciferyl adenylate and pyrophosphate (PPi). This first step requires the prescence of magnesium ions (Osamu 5). Next, the enzyme luciferase speeds up oxidation of luciferyl adenylate to form oxyluciferin, adenosine phosphate (AMP), and light. This two-part process can be expressed as:

Luceferin + ATP → Luciferyl adenylate + PPi

Luciferyl adenalte + O2 → Oxyluciferin + CO2 + AMP + Light

FIREFLY CONTROL

Although the reaction has been studied, the methods firefly use to control these flashes is still not well understood. Fireflies release the neurotransmitter octopamine that triggers a luciferin-luciferase reaction within the firefly lantern structure. However neurons synapse on tracheolar cells and not on firefly photocytes. Thus, there is a 17 micrometer gap between tracheolar cells and the photocytes. When fireflies were placed in a dark observation chamber with a steady flow of NO gas at 70 parts per million, adult Photuris fireflies immediately started to flash (Trimmer et al 2).

Another not complexity in firefly light production is that fireflies can display different wavelengths of light. Because their luciferin molecules remain the same, scientists proposed that it was the color variation was the result of changes in the size of the luciferase protein cavity. Theoretically, a larger cavity would allow for more energy loss and thus lower-energy red light. Conversely, a smaller cavity would reduce energy loss and allow for higher-energy yellow and green light. Though this explanation seems to be logical, recent studies published by a team of scientists in Beijing suggest that the color of firefly light is affected by the polarity within the lantern microenvironment. Ya Jun Liu of the Beijing Normal University reports, “We’ve shown that the light wavelength [of the Luciola cruciata firefly] does not depend on the rigid or loose structure of luciferase but on the water H-bond network inside the cavity…Mutations of luciferase on residues involved in this network should modulate the color” (Zyga 2). Though a little light is shed on how fireflies may actually the color of their light, clearly further research on the exact process is required.

Marine Control

In the marine environment, equally diverse techniques are employed to regulate bioluminescence. However, because most marine creatures house bacterial symbionts, light is constantly being produced and is difficult in a sense to turn on or off. Although light can attract prey and is useful in underwater communication, it also attracts unwanted attention from predators. The flashlight fish uses a retractable fold of skin as a shutter to conceal its bright photophore that lies below its eyes. In a similar sense, various luminous squids expand their chromatophores (color pigments) as to block off the emission of light. However, just recently scientists have discovered the first case of bioluminescence controlled by the use of hormones. Unlike the firefly luminescence, the velvet-belly lantern shark Etmopterus spinax relies on hormones to regulate luminescence, not nerves. Etmopterus spinax is now known to melatonin, prolactin, and alpha-MSH. These hormones are not new to science; they’ve been known to control skin coloration in sharks and their cartilgenous relatives. Melatonin yields a slow, long-lasting glow on the underbelly of the shark, supposedly serving as counterilumination. On the other hand, prolactin elecits a quicker shine that only lasts up to an hour. Scientists hypothesize that the Etmopterus spinax use these relatively fast shines to singal to mates. Lastly, alpha-MSH inhibits luminescence. Melatonin is produced by the pineal gland and is known as the “dark hormone” for its role in sleep patterns in animals. As the shark descends deeper into the water column, its pineal gland senses the increasingly dark environment and compensates by producing melatonin. Therefore, melatonin is an ideal regulator because it is linked to the established biological processes of Etmopterus spinax.

Quality of Light

Chemiluminescence is the production of light through chemical reactions. Bioluminescence is simply chemiluminescence by a living organism. Therefore, bioluminescence can be considered to be a subset of chemiluminescence. The chemical reactions used to produce light are extremely efficient when compared to other methods of light production. Hence, bioluminescence is also known as “cold light” due to the relatively low amounts of heat that are produced. Conversely in incandescence, most of the energy used to create light is wasted as heat, and is accordingly dubbed “hot light” (Simon 13). Fluorescence on the other hand is the result of a special coating on the bulb that absorbs ultraviolet energy and then emits the energy as a longer wavelength of visible light. Phosphorescence is similar to fluorescence, but takes more time to re-emit light and is an overall slower process (Binger 1).

INNOVATIONS

The chemical luminol valued for its use as a chemiluminescent detector in crime scene investigations. Forensic investigators use luminol to specifically detect trace amounts of blood at crime scenes. To produce light, luminol needs to be activated by an oxidant. Often, a solution of hydrogen peroxide and a hydroxide salt is used to activate the luminol (Harrison 1). When a solution of luminol and the activator is sprayed upon a crime scene, trace amounts of iron present in the blood serves as a catalyst and speeds up the decomposition of hydrogen peroxide. The products of this chemical reaction are hydrogen and water. The luminol reacts with the hydroxide salt to form a dianion. The oxygen (produced by the decomposition of hydrogen peroxide) then reacts with the diananion to form organic peroxide. This compound is unstable and immediately decomposes to produce 5-aminophthalic acid. Electrons of the 5-aminophthalic acid are initially in an excited state, but they soon return to their ground state and release their excess energy as visible photons. Thus, a blue glow is generated and lasts for up to 30 seconds. Although this technique requires a fairly dark atmosphere, the glow can be recorded by a long-exposure photograph.

Bioluminescence is finding its unique applications in many fields. A team of researchers headed by Ohio State University have discovered how to manipulate a firefly gene to fight a form of cancer. These researchers were hoping to find a way to fight the cancer adult T-cell lymphoma and leukemia (ATLL). Laboratory mice had ATLL tumor cells injected into their abdomens. Normally, the tumor would progress unnoticed until it reached its later more serious phase. However, the ATLL tumor cells were genetically modified to produce firefly luciferase. Upon receiving the altered ATLL cells, the mice were injected with luciferin. This immediately triggered the biochemical reaction characteristic of the firefly, allowing the researchers to clearly record the visual progression of the tumor. Using this precise method of tracking the tumor, the were able to discover that the drug PS-341 killed over 95% of the cancerous cells (“Firefly Genes” 1).